The Effects And Mechanisms Of MAT2A/2B In Porcine Intramuscular Adipogenesis

In animal husbandry,intramuscular fat(IMF)content has been demonstrated as one of the crucial factors of meat quality characteristics,such as tenderness,juiciness and flavor level.Previous study showed that,energy,protein,fatty acids,vitamins and minerals can improve the intramuscular fat content.Recent years,the genes regulate the intramuscular fat deposition have already started to be explored.The adipogenesis is a highly coordinated process that is not only affected by numerous transcription factors and secreted factors,but signals parthway and DNA or histone methylation can also implicate it.So,it is critically important to fully understand the molecular mechanisms of genes in the intramuscular adipogenesis and pork quality improvement.L-met was catalyzed by methionine adenosyltransferase(MAT)and ATP to form S-adenosylmethionine(AdoMet,SAMe),the principal biological methyl donor.MAT2 A encodes for α2 catalytic subunit that interacts with β regulatory subunit(MAT2B encodes)together to regulate the activity of MATII.Previous reports about the function of MAT2 A and MAT2 B gene were mainly focused in the oncocytes.Recently,the role of S-adenosylmethionine in the adipogenesis has also been investigated.Porcine methionine adenosyltransferase2B(MAT2B)gene was identified as a candidate gene in intramuscular fat(IMF)deposition by transcriptome scan using Affymetrix Genechip,and further study found that MAT2 B gene is differentially expressed in skeletal muscle and subcutaneous adipose tissue of obese and lean pigs.However,the function and mechanisms of MAT2 A and MAT2 B affect porcine intramuscular adipogenesis are still not well understood.To investigate the potential role of MAT2 A and MAT2 B during adipocyte differentiation,we monitored their expression after adipogenic induction.Then we constructed MAT2 A and MAT2 B expression and interference vector to overexpress or inhibit their expression level in porcine intramuscular adipocytes.We evaluated the role of MAT2 A and MAT2 B in morphology and gene expression level during differentiation of porcine intramuscular preadipocytes that were examined by Oil Red O staining,real-time qPCR and Western blotting analysis.Next,HPLC,the specific inhibitor of PI3K/AKT signaling(LY294002),RNA-seq and Co-IP experiments were adopted to explore the mechanisms of MAT2 A and MAT2 B on the porcine intramuscular preadipocyte differentiation,which provide strong evidence for exploring the molecular mechanism of MAT2A/2B in porcine intramuscular adipogenesis.The main research results are as follows:1.Porcine preadipocytes were isolated by differential adhesion method.The expression of MAT2 A and MAT2 B gradually increased during porcine preadipocyte differentiation and MAT2 B was highly expressed in the mature adipocytes.2.MAT2 A and MAT2 B recombinant adenovirus vector were constructed and MAT2A/2B expression were remarkably increased in porcine adipocytes after infected with Ad-MAT2 A and Ad-MAT2 B.Lentivirus-mediated MAT2 A and MAT2 B interference vector were constructed and their expression were significantly inhibited in porcine adipocytes after infected with sh-MAT2 A and sh-MAT2 B.3.Overexpresson of MAT2 A and MAT2 B promoted porcine intramuscular adipogenesis and interference of MAT2 A and MAT2 B inhibit aidpogenesis.MATII selective inhibitor – cycloleucine also inhibited intramuscular preadipocyte differentiation.4.RNA-sequencing was conducted to detect the genes expression after MAT2 A interference.We found 374 upregulated genes and 268 downregulated genes(|Log2fold change|>1,p-Value<0.05)and Wnts genes have a significant change.Overexpression of MAT2 A inhibited the expression of DLK1,Wnt1,Wnt3 a and Wnt10 b,but interference of MAT2 A or cycloleucine supplemention promoted the expression of above genes,which were indentified by real-time q PCR and Western blotting.5.MAT2 A promoted the level of H3K27me3 and H3K27 trimethyltransferase – Ezh2;Further analysis found that MAT2 A form a protein complex with Ezh2 to upregulate the H3K27me3 level in Wnt10 b gene locus by Co-immunoprecipitation and Chip-qPCR in porcine intramuscular adipocytes.6.MAT2 B upreulates intracellular S-adenosylmethionine(SAMe)levels to influence porcine intramuscular preadipocyte differentiation.Supplementation with SAMe can partially recover the inhibition effect by MAT2 B shRNA that was identified by EdU lable and Flow cytometric;MAT2B acted on AKT/ERK signal pathway to promote porcine preadipocyte differentiation,MAT2 B interacted with AKT in procine intramuscular preadipocytes.Overexpression of MAT2 B can rescue the inhibition of LY294002 on the AKT phosphorylation.The work presented that MAT2 A and MAT2 B are positive regulators in porcine intramuscular preadipocyte differentiation.MAT2 A affected adipogenesis by interacted with Ezh2 to promote the level of H3K27me3 in the Wnt10 b locus and further inhibited the Wnt10 b expression to promote porcine intramuscular preadipocyte differentiation;MAT2B affected adipogenesis by modulating intracellular SAMe levels and activating AKT/ERK signaling pathway.These findings have important implications regarding pork quality improvement.